Method of Cross-Linking Hyaluronic Acid with Divinylsulfone

ABSTRACT

The present invention relates to methods of producing a homogenous hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with divinylsulfone (DVS), said method comprising the steps of (a) providing an alkaline solution of hyaluronic acid, or salt thereof; (b) adding DVS to the solution of step (a), whereby the hyaluronic acid, or salt thereof, is crosslinked with the DVS to form a gel; (c) treating the gel of step (b) with a buffer, wherein the gel swells and forms a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with DVS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/912,894 filed Jun. 7, 2013, which is a continuation of U.S.application Ser. No. 11/719,790 filed May 21, 2007 (now U.S. Pat. No.8,481,080), which is a 35 U.S.C. 371 national application ofPCT/DK2005/000753 filed Nov. 24, 2005, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2004 01824filed Nov. 24, 2004 and U.S. provisional application No. 60/630,620filed Nov. 24, 2004, the contents of which are fully incorporated hereinby reference.

BACKGROUND

The present invention relates to a process for the preparation ofmodified hyaluronic acid (HA), in particular cross-linked HA, for use incosmetic, biomedical and pharmaceutical applications.

Hyaluronic acid (HA) is a natural and linear carbohydrate polymerbelonging to the class of the non-sulfated glycosaminoglycans. It iscomposed of beta-1,3-N-acetyl glucosamine and beta-1,4-glucuronic acidrepeating disaccharide units with a molecular weight (MW) up to 6 MDa.HA is present in hyaline cartilage, synovial joint fluid, and skintissue, both dermis and epidermis. HA may be extracted from naturaltissues including the connective tissue of vertebrates, from the humanumbilical cord and from cocks' combs. However, it is preferred today toprepare it by microbiological methods to minimize the potential risk oftransferring infectious agents, and to increase product uniformity,quality and availability (US2003/0175902, Novozymes).

Numerous roles of HA in the body have been identified. It plays animportant role in the biological organism, as a mechanical support forthe cells of many tissues, such as the skin, tendons, muscles andcartilage. HA is involved in key biological processes, such as themoistening of tissues, and lubrication. It is also suspected of having arole in numerous physiological functions, such as adhesion, development,cell motility, cancer, angiogenesis, and wound healing. Due to theunique physical and biological properties of HA (includingviscoelasticity, biocompatibility, biodegradability), HA is employed ina wide range of current and developing applications within cosmetics,ophthalmology, rheumatology, drug delivery, wound healing and tissueengineering. The use of HA in some of these applications is limited bythe fact that HA is soluble in water at room temperature, i.e. about 20°C., it is rapidly degraded by hyaluronidase in the body, and it isdifficult to process into biomaterials. Cross-linking of HA hastherefore been introduced in order to improve the physical andmechanical properties of HA and its in vivo residence time.

U.S. Pat. No. 4,582,865 (Biomatrix Inc.) describes the preparation ofcross-linked gels of HA, alone or mixed with other hydrophilic polymers,using divinyl sulfone (DVS) as the cross-linking agent. The preparationof a cross-linked HA or salt thereof using a polyfunctional epoxycompound is disclosed in EP 0 161 887 B1. Other bi- or poly-functionalreagents that have been employed to cross-link HA through covalentlinkages include formaldehyde (U.S. Pat. No. 4,713,448, Biomatrix Inc.),polyaziridine (WO 03/089476 A1, Genzyme Corp.), L-aminoacids orL-aminoesters (WO 2004/067575, Biosphere S.P.A.). Carbodiimides havealso been reported for the cross-linking of HA (U.S. Pat. No. 5,017,229,Genzyme Corp.; U.S. Pat. No. 6,013,679, Anika Research, Inc). Total orpartial cross-linked esters of HA with an aliphatic alcohol, and saltsof such partial esters with inorganic or organic bases, are disclosed inU.S. Pat. No. 4,957,744.

SUMMARY OF THE INVENTION

A problem to be solved by the present invention is how to manufacturehyaluronic acid based hydrogels with improved properties, such as higherhomogeneity, increased softness, and/or easier syringeability.

The cross-linked gels produced by the method of the invention show anincreased homogeneity and an increased softness compared to the standardDVS crosslinked HA-hydrogels. The gels resulting from the method of theinstant invention are also easier to inject through a syringe, as shownin the examples.

Accordingly, in a first aspect the invention relates to a method ofproducing a hydrogel comprising hyaluronic acid, or salt thereof,crosslinked with divinylsulfone (DVS), said method comprising the stepsof:

-   (a) providing an alkaline solution of hyaluronic acid, or salt    thereof;-   (b) adding DVS to the solution of step (a), whereby the hyaluronic    acid, or salt thereof, is crosslinked with the DVS to form a gel;-   (c) treating the gel of step (b) with a buffer, wherein the gel    swells and forms a hydrogel comprising hyaluronic acid, or salt    thereof, crosslinked with DVS.

In a second aspect, the invention relates to a hydrogel comprisinghyaluronic acid, or salt thereof, crosslinked with divinylsulfone (DVS),which is sufficiently homogenous to be injected from a 1 ml syringethrough a 27 G ½ needle over a distance of 55 mm at a speed of 12.5mm/min with a stable injection force, which after the initial 40 secondsof the injection and until the syringe is empty, varies no more thanabout 5 Newton (N), preferably no more than about 4 N, more preferably 3N, 2 N, or most preferably no more than about 1 N.

In a third aspect, the invention relates to a composition comprising ahydrogel as defined in the second aspect, and an active ingredient,preferably the active ingredient is a pharmacologically active agent.

A fourth aspect of the invention relates to a pharmaceutical compositioncomprising an effective amount of a hydrogel as defined in the secondaspect, together with a pharmaceutically acceptable carrier, excipientor diluent.

A fifth aspect relates to a pharmaceutical composition comprising aneffective amount of a hydrogel as defined in the second aspect as avehicle, together with a pharmacologically active agent.

A sixth aspect relates to a cosmetic article comprising as an activeingredient an effective amount of a hydrogel as defined in the secondaspect or a composition as defined in any of the third, fourth, or fifthaspects.

In a seventh aspect, the invention relates to a sanitary, medical orsurgical article comprising a hydrogel as defined in the second aspector a composition as defined in any of the third, fourth, or fifthaspects, preferably the article is a diaper, a sanitary towel, asurgical sponge, a wound healing sponge, or a part comprised in a bandaid or other wound dressing material.

An important aspect relates to a medicament capsule or microcapsulecomprising a hydrogel as defined in the second aspect or a compositionas defined in any of the third, fourth, or fifth aspects.

A number of aspects relate to uses of a hydrogel as defined in thesecond aspect or a composition as defined in any of the third, fourth,or fifth aspects, for the manufacture of a medicament for the treatmentof osteoarthritis, cancer, the manufacture of a medicament for anopthalmological treatment, the manufacture of a medicament for thetreatment of a wound, the manufacture of a medicament for angiogenesis,the manufacture of a medicament for the treatment of hair loss orbaldness, the manufacture of a moisturizer or a cosmetic, or in acosmetic treatment.

DEFINITIONS

The term “hyaluronic acid” is used in literature to mean acidicpolysaccharides with different molecular weights constituted by residuesof D-glucuronic and N-acetyl-D-glucosamine acids, which occur naturallyin cell surfaces, in the basic extracellular substances of theconnective tissue of vertebrates, in the synovial fluid of the joints,in the endobulbar fluid of the eye, in human umbilical cord tissue andin cocks' combs.

The term “hyaluronic acid” is in fact usually used as meaning a wholeseries of polysaccharides with alternating residues of D-glucuronic andN-acetyl-D-glucosamine acids with varying molecular weights or even thedegraded fractions of the same, and it would therefore seem more correctto use the plural term of “hyaluronic acids”. The singular term will,however, be used all the same in this description; in addition, theabbreviation “HA” will frequently be used in place of this collectiveterm.

“Hyaluronic acid” is defined herein as an unsulphated glycosaminoglycancomposed of repeating disaccharide units of N-acetylglucosamine (GlcNAc)and glucuronic acid (GlcUA) linked together by alternating beta-1,4 andbeta-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan,hyaluronate, or HA. The terms hyaluronan and hyaluronic acid are usedinterchangeably herein.

Rooster combs are a significant commercial source for hyaluronan.Microorganisms are an alternative source. U.S. Pat. No. 4,801,539discloses a fermentation method for preparing hyaluronic acid involvinga strain of Streptococcus zooepidemicus with reported yields of about3.6 g of hyaluronic acid per liter. European Patent No. EP0694616discloses fermentation processes using an improved strain ofStreptococcus zooepidemicus with reported yields of about 3.5 g ofhyaluronic acid per liter. As disclosed in WO 03/054163 (Novozymes),which is incorporated herein in its entirety, hyaluronic acid or saltsthereof may be recombinantly produced, e.g., in a Gram-positive Bacillushost.

Hyaluronan synthases have been described from vertebrates, bacterialpathogens, and algal viruses (DeAngelis, P. L., 1999, Cell. Mol. LifeSci. 56: 670-682). WO 99/23227 discloses a Group I hyaluronate synthasefrom Streptococcus equisimilis. WO 99/51265 and WO 00/27437 describe aGroup II hyaluronate synthase from Pasturella multocida. Ferretti et al.discloses the hyaluronan synthase operon of Streptococcus pyogenes,which is composed of three genes, hasA, hasB, and hasC, that encodehyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucosepyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98,4658-4663, 2001). WO 99/51265 describes a nucleic acid segment having acoding region for a Streptococcus equisimilis hyaluronan synthase.

Since the hyaluronan of a recombinant Bacillus cell is expresseddirectly to the culture medium, a simple process may be used to isolatethe hyaluronan from the culture medium. First, the Bacillus cells andcellular debris are physically removed from the culture medium. Theculture medium may be diluted first, if desired, to reduce the viscosityof the medium. Many methods are known to those skilled in the art forremoving cells from culture medium, such as centrifugation ormicrofiltration. If desired, the remaining supernatant may then befiltered, such as by ultrafiltration, to concentrate and remove smallmolecule contaminants from the hyaluronan. Following removal of thecells and cellular debris, a simple precipitation of the hyaluronan fromthe medium is performed by known mechanisms. Salt, alcohol, orcombinations of salt and alcohol may be used to precipitate thehyaluronan from the filtrate. Once reduced to a precipitate, thehyaluronan can be easily isolated from the solution by physical means.The hyaluronan may be dried or concentrated from the filtrate solutionby using evaporative techniques known to the art, such as lyophilizationor spraydrying.

Host Cells

A preferred embodiment relates to the method of the first aspect,wherein the hyaluronic acid or salt thereof is recombinantly produced,preferably by a Gram-positive bacterium or host cell, more preferably bya bacterium of the genus Bacillus.

The host cell may be any Bacillus cell suitable for recombinantproduction of hyaluronic acid. The Bacillus host cell may be a wild-typeBacillus cell or a mutant thereof. Bacillus cells useful in the practiceof the present invention include, but are not limited to, Bacillusagaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.Mutant Bacillus subtilis cells particularly adapted for recombinantexpression are described in WO 98/22598. Non-encapsulating Bacilluscells are particularly useful in the present invention.

In a preferred embodiment, the Bacillus host cell is a Bacillusamyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. Ina more preferred embodiment, the Bacillus cell is a Bacillusamyloliquefaciens cell. In another more preferred embodiment, theBacillus cell is a Bacillus clausii cell. In another more preferredembodiment, the Bacillus cell is a Bacillus lentus cell. In another morepreferred embodiment, the Bacillus cell is a Bacillus licheniformiscell. In another more preferred embodiment, the Bacillus cell is aBacillus subtilis cell. In a most preferred embodiment, the Bacillushost cell is Bacillus subtilis A164Δ5 (see U.S. Pat. No. 5,891,701) orBacillus subtilis 168Δ4.

Molecular Weight

The content of hyaluronic acid may be determined according to themodified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4:330-334). Moreover, the average molecular weight of the hyaluronic acidmay be determined using standard methods in the art, such as thosedescribed by Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt,1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, “LightScattering University DAWN Course Manual” and “DAWN EOS Manual” WyattTechnology Corporation, Santa Barbara, Calif.

In a preferred embodiment, the hyaluronic acid, or salt thereof, of thepresent invention has a molecular weight of about 10,000 to about10,000,000 Da. In a more preferred embodiment it has a molecular weightof about 25,000 to about 5,000,000 Da. In a most preferred embodiment,the hyaluronic acid has a molecular weight of about 50,000 to about3,000,000 Da.

In a preferred embodiment, the hyaluronic acid or salt thereof has amolecular weight in the range of between 300,000 and 3,000,000;preferably in the range of between 400,000 and 2,500,000; morepreferably in the range of between 500,000 and 2,000,000; and mostpreferably in the range of between 600,000 and 1,800,000.

In yet another preferred embodiment, the hyaluronic acid or salt thereofhas a low average molecular weight in the range of between 10,000 and800,000 Da; preferably in the range of between 20,000 and 600,000 Da;more preferably in the range of between 30,000 and 500,000 Da; even morepreferably in the range of between 40,000 and 400,000 Da; and mostpreferably in the range of between 50,000 and 300,000 Da.

Salts and Crosslinked HA

A preferred embodiment relates to a method of the first aspect, whichcomprises an inorganic salt of hyaluronic acid, preferably sodiumhyaluronate, potassium hyaluronate, ammonium hyaluronate, calciumhyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalthyaluronate.

Other Ingredients

In a preferred embodiment, the product produced by the method of theinvention may also comprise other ingredients, preferably one or moreactive ingredient, preferably one or more pharmacologically activesubstance, and also preferably a water-soluble excipient, such aslactose or a non-biologically derived sugar.

Non-limiting examples of an active ingredient or pharmacologicallyactive substance which may be used in the present invention includevitamin(s), protein and/or peptide drugs, such as, human growth hormone,bovine growth hormone, porcine growth hormone, growth hormone releasinghormone/peptide, granulocyte-colony stimulating factor, granulocytemacrophage-colony stimulating factor, macrophage-colony stimulatingfactor, erythropoietin, bone morphogenic protein, interferon orderivative thereof, insulin or derivative thereof, atriopeptin-III,monoclonal antibody, tumor necrosis factor, macrophage activatingfactor, interleukin, tumor degenerating factor, insulin-like growthfactor, epidermal growth factor, tissue plasminogen activator, factorIIV, factor IIIV, and urokinase.

A water-soluble excipient may be included for the purpose of stabilizingthe active ingredient(s), such excipient may include a protein, e.g.,albumin or gelatin; an amino acid, such as glycine, alanine, glutamicacid, arginine, lysine and a salt thereof; carbohydrate such as glucose,lactose, xylose, galactose, fructose, maltose, saccharose, dextran,mannitol, sorbitol, trehalose and chondroitin sulphate; an inorganicsalt such as phosphate; a surfactant such as TWEEN® (ICI), poly ethyleneglycol, and a mixture thereof. The excipient or stabilizer may be usedin an amount ranging from 0.001 to 99% by weight of the product.

Several aspects of the invention relate to various compositions andpharmaceuticals comprising, among other constituents, an effectiveamount of the crosslinked HA product, and an active ingredient,preferably the active ingredient is a pharmacologically active agent; apharmaceutically acceptable carrier, excipient or diluent, preferably awater-soluble excipient, and most preferably lactose.

A preferred embodiment of the invention relates to products orcompositions of the invention comprised in an effervescent tablet, whichmay otherwise be formulated as described in the art. For instance, aneffervescent tablet may comprise citric acid, sodium bicarbonate, and anoligosaccharide or other sugar. Effervescent tablets are easy to store,and with the fast-dissolving product of the present invention, they arequickly dissolved and thus provide an ideal means of oraladministration.

In addition, aspects of the invention relate to articles comprising aproduct as defined in the first aspect or a composition as defined inthe aspects and embodiments above, e.g., a cosmetic article, a sanitaryarticle, a medical or surgical article. In a final aspect the inventionrelates to a medicament capsule or microcapsule comprising a product asdefined in the first aspect or a composition as defined in other aspectsand embodiments of the invention.

FIGURES

FIG. 1 illustrates the weight loss of DVS-HA hydrogels resulting fromHyaluronidase degradation as a function of time. DVS-HA hydrogelsprepared with a heating step (Heated), as described in Example 2, werecompared to DVS-HA hydrogels which had not been heat treated (Notheated′).

FIG. 2 shows the time course of the pH-value of a set of DVS crosslinkedHA hydrogels with different ratios or degrees of crosslinking, duringswelling in phosphate buffer (pH=7.0), as described in detail in Example6 below.

FIG. 3 shows the elastic modulus (G′), labelled with a circle, and theshear loss or viscous modulus (G″), labelled with a square, of two HAhydrogels prepared according to the invention, one prepared with aHA/DVS ratio of 10:1 and 6% HA, and the other with a HA/DVS ratio of15:1 and 6% HA, as described in detail in example 7 below. The elasticmodulus (G′: circle) of the HA/DVS 10:1 hydrogel is the upper line(y-axis) at all frequencies (x-axis), and the shear loss modulus (G″:square) of the HA/DVS 10:1 hydrogel is the lower line, except at theextreme left-hand side of the x-axis, where it is the upper line.

FIG. 4 shows the syringeability of DVS cross-linked HA hydrogels (HA/DVS10:1, wt) prepared following the process described in example 2 herein(′new, heated′), and a hydrogel prepared according to the prior art (seeU.S. Pat. No. 4,582,865, example 1) without heating, as described inexample 9 below. The y-axis shows the injection force in Newton,beginning at 0.0 with increments of 2.5, and ending at 35.0.

FIG. 5 shows the syringeability of DVS cross-linked HA hydrogels (HA/DVS15:1, wt) prepared following the process described in example 2 herein(new, heated′), and a hydrogel prepared according to the prior art (seeU.S. Pat. No. 4,582,865, example 1) without heating, as described inexample 9 below. The y-axis shows the injection force in Newton,beginning at 0.0 with increments of 2.5, and ending at 30.0.

DETAILED DESCRIPTION OF THE INVENTION

The first aspect of the invention relates to a method of producing ahydrogel comprising hyaluronic acid, or salt thereof, crosslinked withdivinylsulfone (DVS), said method comprising the steps of:

-   (a) providing an alkaline solution of hyaluronic acid, or salt    thereof;-   (b) adding DVS to the solution of step (a), whereby the hyaluronic    acid, or salt thereof, is crosslinked with the DVS to form a gel;-   (c) treating the gel of step (b) with a buffer, wherein the gel    swells and forms a hydrogel comprising hyaluronic acid, or salt    thereof, crosslinked with DVS.

It has previously been described how to produce hyaluronic acidrecombinantly in a Bacillus host cell, see WO 2003/054163, Novozymes NS,which is incorporated herein in its entirety.

Accordingly, in a preferred embodiment, the invention relates to themethod of the first aspect, wherein the hyaluronic acid, or saltthereof, is recombinantly produced in a Bacillus host cell.

Various molecular weight fractions of hyaluronic acid have beendescribed as advantageous for specific purposes.

A preferred embodiment of the invention relates to a method of the firstaspect, wherein the hyaluronic acid, or salt thereof, has an averagemolecular weight of between 100 and 3,000 kDa, preferably between 500and 2,000 kDa, and most preferably between 700 and 1,800 kDa.

The initial concentration of hyaluronic acid, or a salt thereof, in themethod of the invention, influences the properties of the resultingcrosslinked gel, and of the swollen hydrogel.

Therefore, a preferred embodiment of the invention relates to a methodof the first aspect, wherein the alkaline solution comprises dissolvedhyaluronic acid, or salt thereof, in a concentration of between 0.1%-40%(w/v).

The pH value during the crosslinking reaction also influences theoutcome, so in a preferred embodiment the invention relates to a methodof the first aspect, wherein the alkaline solution comprises dissolvedsodium hydroxide in a concentration of between 0.001-2.0 M.

It is also noteworthy that the concentration of the crosslinking agenthas a profound impact on the resulting gels.

Consequently, a preferred embodiment of the invention relates to amethod of the first aspect, wherein DVS is added to the solution of step(a) in a weight ratio of between 1:1 and 100:1 of HA/DVS (dry weight),preferably between 2:1 and 50:1 of HA/DVS (dry weight).

The inventors found that an initial period of stirring during and/orimmediately after adding the DVS to the HA-solution was desirable toachieve satisfactory gelling.

Accordingly, a preferred embodiment of the invention relates to a methodof the first aspect, wherein DVS is added with stirring to the solutionof step (a), and wherein the solution temperature is maintained in therange of 5° C.-50° C., preferably in the range of 15° C.-40° C., morepreferably in the range of 20° C.-30° C.; preferably the stirring iscontinued for a period of between 1-180 minutes.

In another preferred embodiment of the method of the first aspect, theDVS is added without stirring to the solution of step (a).

The present inventors determined that a heating step was beneficialafter addition of the DVS to the solution.

Accordingly, a preferred embodiment of the invention relates to a methodof the first aspect, wherein the solution temperature in step (b) isheated to a temperature in the range of 20° C.-100° C., preferably inthe range of 25° C.-80° C., more preferably in the range of 30° C.-60°C., and most preferably in the range of 35° C.-55° C., and wherein thetemperature is maintained in this range for a period of at least 5minutes, preferably at least 10 minutes, 20 minutes, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or most preferably atleast 180 minutes; preferably without stirring.

It is advantageous to leave the gel standing at room temperature for abrief period after the crosslinking reaction has taken place.

In a preferred embodiment of the method of the first aspect, the gel ismaintained for a period of at least 5 minutes, preferably at least 10minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, or most preferably at least 180 minutes, at atemperature in the range of 0° C.-40° C., preferably in the range of 10°C.-30° C.

Many types of buffers, as are well known to the skilled person, havebeen envisioned as suitable for the swelling and neutralizing of thecrosslinked gel of the invention. In a preferred embodiment the buffercomprises a buffer with a pH value in the range of 2.0-8.0, preferablyin the range of 5.0-7.5.

Optimally, a suitable buffer is chosen with a pH value, which results inthat the swollen hydrogel has a pH value as close to neutral aspossible. In a preferred embodiment, the buffer comprises a buffer witha pH value, which results in that the hydrogel has a pH value between5.0 and 7.5.

It is preferred that the buffer in the method of the first aspectcomprises a phosphate buffer and/or a saline buffer.

In the swelling step the buffer must have a sufficient volume for it toaccommodate the swelling gel until the gel is fully swollen.Accordingly, in a preferred embodiment of the method of the firstaspect, the buffer in step (c) has a volume of at least 3 times thevolume of the gel of step (b).

In a preferred embodiment of the method of the first aspect, theswelling in step (c) is carried out at a temperature of between 20°C.-50° C. for a period of at least 5 minutes, preferably at least 10minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, or most preferably at least 180 minutes.

It is also a preferred that the hydrogel formed in step (c) is washed atleast once with water, water and a phosphate buffer, water and a salinebuffer, or water and a phosphate buffer and a saline buffer, with a pHvalue in the range of of 2.0-8.0, preferably in the range of 5.0-7.5.

EXAMPLES Example 1 Preparation of DVS-Crosslinked HA Hydrogels

This example illustrates the preparation of DVS-cross-linked HAhydrogels with concomitant swelling and pH adjustment.

Sodium hyaluronate (HA, 770 kDa, 1 g) was dissolved into 0.2M NaOH togive a 4% (w/v) solution, which was stirred at room temperature, i.e.about 20° C., for 1 h. Three replicates were prepared. Divinylsulfone(DVS) was then added to the HA solutions in sufficient amount to giveHA/DVS weight ratios of 10:1, 7:1, and 5:1, respectively. The mixtureswere stirred at room temperature for 5 min and then allowed to stand atroom temperature for 1 h. The gels were then swollen in 160 mL phosphatebuffer (pH 4.5 or 6.5) for 24 h, as indicated in Table 1.

TABLE 1 Conditions for DVS-HA hydrogel preparation. Gel ID HA/DVS weightratio Phosphate buffer used for swelling 1  5:1 160 ml (pH 4.5) 2  7:180 ml (pH 4.5) + 80 ml (pH 6.5) 3 10:1 160 ml (pH 6.5)

The pH of the gels was stabilized during the swelling step. Afterswelling, any excess buffer was removed by filtration and the hydrogelswere briefly homogenized with an IKA® ULTRA-TURRAX® T25 homogenizer (IkaLabortechnik, DE). The volume and pH of the gels were measured (seeTable 2).

TABLE 2 Characteristics of DVS-HA hydrogels. HA/ Volume DVS of HA weightswollen concentration Gel ID ratio gel (w/v) pH Appearance Softness 15:1 70 mL 1.4% 7.1 Transparent, + homogenous 2 7:1 70 mL 1.4% 7.6Transparent, ++ homogenous 3 10:1  70 mL 1.4% 7.5 Transparent, +++homogenous

The pH of the hydrogels ranged from 7.1 to 7.6 (table 2), which confirmsthat the swelling step can be utilized to adjust the pH in this process.All the hydrogels occupied a volume of 70 mL, which corresponds to a HAconcentration of ca. 1.4% (w/v). They were transparent, coherent andhomogenous. Softness increased with decreasing cross-linking degree(Table 2).

Example 2 Preparation of Homogenous DVS-Crosslinked HA Hydrogels

This example illustrates the preparation of highly homogenousDVS-cross-linked HA hydrogels.

Sodium hyaluronate (770 kDa, 2 g) was dissolved into 0.2M NaOH withstirring for approx. 1 hour at room temperature to give a 8% (w/v)solution. DVS was then added so that the HA/DVS weight ratio was 7:1.After stirring at room temperature for 5 min, one of the samples washeat treated at 50° C. for 2 h without stirring, and then allowed tostand at room temperature overnight. The resulting cross-linked gel wasswollen into 200 ml phosphate buffer (pH 5.5) 37° C. for 42 or 55 h, andfinally washed twice with 100 ml water, which was discarded. Volume andpH were measured, as well as the pressure force necessary to push thegels through a 27G*½ injection needle (see Table 3).

TABLE 3 Characteristics of DVS-cross-linked HA hydrogels. Stability ofVolume HA pressure Heat of concen- force Gel treat- swollen trationSoft- during ID ed gel (w/v) pH Appearance ness injection 1 Yes 145 mL1.4% 6.1 Transparent, +++ +++ homogenous 2 No  90 mL 1.1% 6.7Transparent, + + homogenousThe cross-linked HA hydrogel prepared according to this exampleexhibited a higher swelling ratio and an increased softness compared toa control hydrogel which was not heat treated (Table 3). The pressureforce applied during injection through a 27G*½ needle was more stablethan that of the latter sample, indicating that the cross-linked HAhydrogel is more homogenous.

Example 3 Biostability of DVS-Crosslinked HA Hydrogels

This example illustrates the in vitro biostability of DVS-cross-linkedHA hydrogels using enzymatic degradation.

A bovine testes hyaluronidase (HAase) solution (100 U/mL) was preparedin 30 mM citric acid, 150 mM Na₂HPO₄, and 150 mM NaCl (pH 6.3). DVS-HAcross-linked hydrogel samples (ca. 1 mL) were placed into safe-lockglass vials, freeze-dried, and weighed (W₀; Formula 1). The enzymesolution (4 mL, 400 U) was then added to each sample and the vials wereincubated at 37° C. under gentle shaking (100-200 rpm). At predeterminedtime intervals, the supernatant was removed and the samples were washedthoroughly with distilled water to remove residual salts, they were thenfreeze-dried, and finally weighed (W_(t); Formula 1).

The biodegradation is expressed as the ratio of weight loss to theinitial weight of the sample (Formula 1). Weight loss was calculatedfrom the decrease of weight of each sample before and after theenzymatic degradation test. Each biodegradation experiment was repeatedthree times.

Formula 1:

${{Weightloss}(\%)} = {\frac{{W\; 0} - {Wt}}{W\; 0} \times 100}$

The results are shown in table 4, as well as in FIG. 1, whichillustrates the weight loss of DVS-HA hydrogels resulting from HAasedegradation as a function of time. DVS-HA hydrogels prepared asdescribed in example 2 (‘Heated’) were compared to DVS-HA hydrogelswhich had not been heat treated (‘Not heated’). For both types of gel,degradation was fast during the first four hours, and then proceededslower until completion at 24 h. Importantly there was a significantvariation of the weight loss values for the samples which had not beenheated as compared to the hydrogel prepared with a heating step asdescribed in example 2. This clearly illustrates that a highlyhomogenous DVS-cross-linked HA hydrogel is obtained by using the processdescribed in example 2.

Example 4 Preparation of Water-in-Oil Emulsions for Cosmetics

In this and in the following example, DVS-crosslinked HA hydrogels wereformulated into creams and serums, that when applied to the skinincrease the skin moisturization and elasticity, and provide immediateanti-aging effect, as well as film-forming effect

A typical formulation of a water-in-oil (w/o) emulsion containing 2%DVS-cross-linked HA. Each phase (A to E) was prepared separately bymixing the defined ingredients (see Table 4). Phase B was then added tophase A under stirring with a mechanical propel stirring device and at atemperature less than 40° C. Phase C was then added followed by phase Dand finally phase E under stirring. Formulations were also made, whereinthe HA hydrogel concentration was 4%, 6% and 8%, respectively, in PhaseD, to give a range of w/o formulations.

TABLE 4 Proportion Phase Ingredient (w/w) Function A Cyclopentasiloxane,dimethicone 10%  Emollient Cyclopentasiloxane 15%  EmollientCyclopentasiloxane and PEG/PPG- 4% Emulsifier 20/15 DimethiconeHydrogenated polydecene 8% Emollient B Water 49.3%   Sodium chloride0.2%   C Tocopheryl acetate 0.5%   Antioxidant D DVS Cross-linked sodium2% hyaluronate Water 10%  E Phenoxyethanol, ethylhexylglycerin 1%Preservative

Another typical formulation of a w/o-emulsion containing 2%DVS-crosslinked HA is shown in table 5. Each phase (A to F) in table 5was prepared separately by mixing the defined ingredients (see Table 5).Phase B was mixed with phase A and the resulting oil phase was heated at75° C. Phase C was also heated to 75° C. The oil phase was added tophase C at 75° C. under stirring with a mechanical propel stirringdevice. The emulsion was then cooled down to less than 40° C., afterwhich phase D was added, followed by phase E and finally phase F understirring. Formulations were also made, wherein the HA hydrogelconcentration was 4%, 6% and 8%, respectively, in Phase E, to give arange of w/o formulations.

TABLE 5 Proportion Phase Ingredient (w/w) Function A Hydrogenatedpolydecene 18%  Emollient Acrylates/C10-30 alkyl acrylate 1% Thickenercrosspolymer B Sodium cocoyl Glutamate 10%  Emulsifier C Aqua 53.5%  Distarch Phosphate 2% Texture agent D Tocopheryl acetate 0.5%  Antioxidant Cyclopentasiloxane, dimethicone 2% Feeling and spreadingagent E Cross-linked sodium 2% hyaluronate Aqua 10%  F Phenoxyethanol,ethylhexylglycerin 1% Preservative

Example 5 Preparation of Silicone Serums

A typical formulation of a silicone serum containing 2% DVS-cross-linkedHA was prepared as shown in table 6. All ingredients were mixed at thesame time under very high stirring and at less than 40° C. (see table6). Formulations were also prepared, wherein the HA hydrogelconcentration was 4%, 6% and 8%, respectively, to give a range ofserums.

TABLE 6 Proportion Ingredient (w/w) Function Cyclopentasiloxane 60% Line blurring effect, C30-45 Alkyl Cetearyl Dimethicone thickener,vehicle Crosspolymer Cyclopentasiloxane 34.5%   Vehicle, emollientPolymethylsilsesquioxane 2.5%   Soft powdery feel Cross-linked sodium 2%hyaluronate Phenoxyethanol, ethylhexylglycerin 1% Preservative

Example 6 pH Equilibration During Swelling; a Kinetics Study

A kinetics study showed that DVS cross-linked HA hydrogels with neutralpH are obtained after swelling in phosphate buffer (pH 7.0) for 8 to 14hours, depending on the degree of cross-linking. A set of DVScross-linked HA hydrogels was prepared as described in the above, usingfrom 4 to 8% HA solution, and using various amounts of DVS cross-linker,as indicated in Table 7.

TABLE 7 Initial HA concentration HA/DVS weight Entry (w/v) ratio 1 4%2.5:1  2 6% 15:1 3 8% 15:1 4 6% 10:1

At regular intervals (every 2 hours), the hydrogels were removed duringthe heat-treatment and decanted, and pH was measured (see FIG. 2). Freshswelling buffer was used after each measurement. The results show that,for all hydrogels, pH ranged between 11 and 12 after 2-hours ofswelling. Then pH gradually decreased to 7.2-7.5.

The decrease was faster for the hydrogels that were less cross-linked,i.e., where the HA/DVS-ratio was higher. The decrease in pH is shown forthe HA 6% solution and two different ratios of HA/DVS in FIG. 2, wherethe HA/DVS ratio of 10:1 is labelled with triangles, and 15:1 islabelled with squares. In these two cases, pH was neutralized within 8hours. In contrast, neutral pH was reached after 14 hour-swelling forhydrogels with either a higher HA concentration (e.g. 8%) or a higherdegree of cross-linking (e.g. HA/DVS ratio of 2.5). These observationsare in accordance with the fact that HA molecules in the lowcross-linked hydrogels exhibit greater freedom and flexibility, allowinggood hydration and thereby faster pH equilibration.

Example 7 Visco Elastic Properties of Hydrogels Based on DVS-CrosslinkedHA

The rheological measurements were performed on a Physica MCR 301rheometer (Anton Paar, Ostfildern, Germany) using a plate-plate geometryand at a controlled temperature of 25° C. The visco-elastic behavior ofthe samples was investigated by dynamic amplitude shear oscillatorytests, in which the material was subjected to a sinusoidal shear strain.First, strain/amplitude sweep experiments were performed to evaluate theregion of deformation in which the linear viscoelasticity is valid. Thestrain typically ranged from 0.01 to 200% and the frequency was set to 1Hz. Then, in the linear visco-elastic regions, the shear storage modulus(or elastic modulus G′) and the shear loss modulus (or viscous modulus,G″) values were recorded from frequency sweep experiments at a constantshear strain (10%) and at a frequency between 0.1 and 10 Hz. Thegeometry, the NF and the gap were PP 25, 2 and 1 mm, respectively.

G′ gives information about the elasticity or the energy stored in thematerial during deformation, whereas G″ describes the viscous characteror the energy dissipated as heat. In particular, the elastic modulusgives information about the capability of the sample to sustain load andreturn in the initial configuration after an imposed stress ordeformation. In all experiments, each sample was measured at least threetimes.

The results (FIG. 3) showed that for both hydrogels:

-   -   G′>G″ and    -   G′ is ALMOST independent of the frequency.

In case of the hydrogel with a higher degree of cross-linking (i.e.lower HA/DVS ratio: 10/1) G′ is one order of magnitude higher than G″,indicating that this sample behaves as a strong gel material. Briefly,the overall rheological response is due to the contributions of physicaland chemical crosslinks, and to topological interactions among the HAmacromolecules. The interactions among the chains bring about areduction of their intrinsic mobility that is not able to releasestress, and consequently the material behaves as a three-dimensionalnetwork, where the principal mode of accommodation of the applied stressis by network deformation. Moreover, this hydrogel was more elastic thanthat with a lower degree of cross-linking (i.e. higher ratio of HA/DVS:15:1). Indeed, the higher the number of permanent covalent cross-links,the larger the number of entanglements, and therefore the higher theelastic response of the hydrogel.

Example 8 Network Structural Parameters

In this experiment, the viscoelastic properties were evaluated on arotational rheometer (Gemini, Bohlin Instruments, UK) using a parallelplate geometry (PP30 cell). The tests were carried out at the controlledtemperature of 25° C. using a thermostatic bath. To avoid waterevaporation, the humidity of the chamber containing the samples wascontrolled by a humidity Control Accessory.

The hydrogels were subjected to periodic oscillation in a dynamicexperiment (small amplitude frequency sweep tests) to evaluate thedependence of the elastic and viscous moduli, G′ and G″. The frequencyrange was 0.01 Hz-10 Hz. In order to identify the linear viscoelasticresponse range of the materials, preliminary strain sweep tests wereperformed on the samples at the oscillation frequency of 1 Hz. The testswere repeated at least three times on each sample.

The values of the elastic modulus can be used to estimate the parameterof the network structure. As G is proportional to the number ofentanglements (Ferry, 1980), the elastic modulus can be expressedthrough:

G≅R·T·z  Formula 2:

Wherein RT is the thermal energy, and z is the number of theentanglement points or cross-linking point expressed in mol/volume. Theparameter z can be calculated by:

Formula 3:

$z \approx \frac{c}{M_{e}}$

Wherein c is the polymer concentration, and M_(e) is the averagemolecular weight of the polymer segments between two entanglements.Substituting in Formula 2, M_(e) can be estimated by the followingequation:

Formula 4:

$M_{e} \cong \frac{R \cdot T \cdot c}{G}$

To calculate G by means of Formula 4, the validity of the rubberelasticity theory was assumed and the temporary network of gel-likematerial was presumed to behave as does vulcanized rubber upon stimulusof a time scale shorter than the life time of the entanglement network(Flory, 1953). The “dangling ends”, which are the polymer chain segmentsattached to the network by only one entanglement point, do notcontribute to the G value because they cannot store elastic energy.Thus, a correction is needed in Formula 4 (Flory, 1953):

Formula 5:

$G \cong {\frac{R \cdot T \cdot c}{M_{d}}\left( {1 - {2\frac{M_{d}}{M_{n}}}} \right)}$

Where Mn is the number average molecular weight. Using the “equivalentnetwork model” (Schurz, 1991), it is possible to estimate DN which isthe average distance between the entanglements points in a idealized“equivalent network”:

$D_{N} = \sqrt[3]{\frac{6 \cdot M_{d}}{\pi \cdot c \cdot A}}$

Formula 6:

Wherein A is Avogadro's number.

The results of D_(N) and M_(d) are reported in table 8. It can benoticed that the higher Md (248120 g/mol) and higher Dn (46 nm) areobtained for sample 1. Sample 2 had the lowest Md (204000) and a Dnvalue of 43.5 nm. Samples 3 and 4, which have the same elastic modulus,are characterized by Md of 240000 g/mol and Dn of 42 nm.

TABLE 8 Network parameters for DVS-HA hydrogels. Initial HA Phosphate HASample conc. HA/DVS buffer G′ concentration Md D_(N) ID (w/v) wt ratioconcentration^(a) [Pa]^(b) (g/l) (g/mol) (nm) 1 4% 2.5:1 50 mM 11 8248120 46 2 6%  10:1 50 mM 27.7 7.8 204000 43.5 3 4% 2.5:1 150 mM 18 10238000 42 4 6%  10:1 150 mM 18 10.7 240700 41.5 ^(a)During swelling;^(b)Value of the elastic modulus at 0.1 Hz.

Example 9 Syringeability of DVS-Crosslinked HA Hydrogels

The syringeability of DVS cross-linked HA hydrogels prepared accordingto the present invention was compared to that of hydrogels preparedaccording to prior art, e.g., as in example 1 of U.S. Pat. No.4,582,865.

The syringeability was measured on a Texture analyzer (Stable MicroSystems, TA. XT Plus) as the force (in N) needed to inject the hydrogelthrough a 27G ½ needle over a distance of 55 mm at a speed of 12.5mm/min. Hydrogel samples were transferred into a 1 mL-syringe fittedwith a 27G ½ needle and the syringe was placed in the holder. Eachsample was measured three times. FIGS. 4 and 5 illustrate thesyringeability of DVS cross-linked HA hydrogels with HA/DVS weightratios of 10:1 and 15:1, respectively.

The injection profiles recorded in FIGS. 4 and 5 are characteristic ofthe sample homogeneity. Indeed, the more stable the applied injectionforce is, the more homogenous the hydrogel is. Moreover, a low forcecorresponds to an easy injection of the hydrogel by the operator.

The results clearly indicated that the DVS HA-hydrogels producedaccording to the process described herein were far more homogenous thanthose obtained from prior art method. Note, that the prior art sampleshad to be homogenized mechanically in order for them to be syringeableat all. This homogenization created small particles, the presence ofwhich lead to very irregular injection profiles.

Furthermore, the cross-linked hydrogels prepared according to thepresent invention were easier to inject through a fine needle, asdemonstrated by the lower force required. It is noteworthy that theinjection force increases with an increasing degree of cross-linking dueto the formation of a stronger network.

Example 10 Formulations of Crosslinked DVS-HA Hydrogels for LocalOphthalmology

A typical formulation of a 500 mL eye-drop solution containing 1% (w/v)DVS-cross-linked HA is shown in table 9. All ingredients were weighedand transferred into a 500 mL volumetric flask. Water (300 mL) was addedand the mixture was stirred at room temperature for 5 h. pH was adjustedto 7.2 with 2M NaOH and the volume was adjusted to exactly 500 mL withmilliQ water.

TABLE 9 Ingredient Amount Function Cross-linked sodium 5 g Lubricanthyaluronate Viscosity enhancer Moisturizer/hydration agent Sodiumethylene diamine tetra 50 mg Chelating agent acetate (EDTA) Sodiumdihydrogen phosphate 20 mg Buffer dihydrate (NaH₂PO₄, 2H₂O) Disodiumhydrogen phosphate 140 mg Buffer dihydrate (Na₂HPO₄, 2H₂O) Sodiumchloride 4.5 g Polyaminopropyl Biguanide 3.25 microL Preservative (PHMB)Milli-Q water Up to 500 mL

Example 11 Crosslinked HA/DVS Hydrogel with Preservative

A DVS-cross-linked HA hydrogel was prepared using 1.5 g of sodium HA in0.2 M NaOH to give a 6% (w/v) solution. The HA/DVS weight ratio was10:1. The hydrogel was prepared in three replicates according to theprocedure described in example 2 until the swelling step, after which itwas treated as follows: After incubation in an oven at 50° C. for twohours, the hydrogel was immersed into Na2HPO4/NaH2PO4 buffer (1 L, 50mM, pH 7.0) containing the preservative(2-phenoxyethanol/3[(2-ethylhexyl)oxy]1,2-propanediol).

The concentration of preservative was 10 mL/mL to target a finalconcentration of 1% (v/v) in the swollen hydrogel. It was anticipatedthat the preservative would diffuse into the hydrogel during theincubation, and that at the same time, microbial contamination in thebuffer would be prevented.

The vessel was covered with parafilm and placed in an oven at 37° C.After 1 h, the swelling bath was removed and the hydrogel was swollen ina fresh phosphate buffer containing 10 mL/mL preservative for 6-7 h.This step was repeated until the swelling time was 12 h, whereafter thepH was measured. Swelling was continued for another 2.5 h to reachneutral pH.

The amount of preservative incorporated into the hydrogel was determinedby UV-spectrophotometry (Thermo Electron, Nicolet, Evolution 900,equipment nr. 246-90). A 1% (v/v) solution of the preservative inphosphate buffer was first analyzed to select the wavelength.Approximately 5 mL of hydrogel were collected using a pipette.Typically, samples were collected in the center of the swollen roundhydrogel, and in the north, east, south, and west “sides” of the roundgel.

The samples were then transferred into a cuvette and the absorbance wasread at 292 nm. Each sample was read three times and the absorbance waszeroed against a blank DVS-cross-linked HA hydrogel, containing nopreservative.

The results showed that the amount of preservative incorporated in theDVS-HA hydrogel ranged between 0.91% and 1.02% (see Table 10). There wasvery good reproducibility between the replicates. Importantly, nosignificant difference between samples from the same hydrogel wasobserved, indicating a homogenous diffusion of the preservative into thehydrogel.

TABLE 10 Amount of incorporated preservative into DVS-HA hydrogel uponswelling in a 1% preservative-spiked phosphate buffer for 14.5 h.Preservative Average Sample Absorbance* concentration concentrationSample ID site (292 nm) (%, v/v) (%, v/v) Replicate Center 0.072 1.020.91 1 Side 0.058 0.82 Side 0.066 0.94 Side 0.057 0.81 Side 0.068 0.97Replicate Middle 0.076 1.08 1.02 2 Side 0.069 0.98 Side 0.082 1.17 Side0.071 1.01 Side 0.062 0.88 Replicate Middle 0.083 1.18 1.02 3 Side 0.0741.05 Side 0.069 0.98 Side 0.066 0.94 Side 0.068 0.97 *The absorbance isthe mean value of three measurements performed on the same sample.

1. A hydrogel comprising hyaluronic acid, or a salt thereof, crosslinkedwith divinylsulfone (DVS), wherein the hydrogel is sufficientlyhomogenous that when injected from a 1 ml syringe through a 27G ½ needleover a distance of 55 mm at a speed of 12.5 mm/min, the injection forcevaries no more than about 5 Newton (N) when measured from 40 secondsafter the initial injection until the syringe is empty.
 2. The hydrogelaccording to claim 1, wherein the injection force varies no more thanabout 3 Newton (N) when measured from 40 seconds after the initialinjection until the syringe is empty.
 3. The hydrogel according to claim1, wherein the injection force varies no more than about 2 Newton (N)when measured from 40 seconds after the initial injection until thesyringe is empty.
 4. The hydrogel according to claim 1, wherein theinjection force varies no more than about 1 Newton (N) when measuredfrom 40 seconds after the initial injection until the syringe is empty.5. The hydrogel according to claim 1, wherein the hyaluronic acid orsalt thereof has a molecular weight in the range of between 300,000 and3,000,000.
 6. The hydrogel according to claim 1, wherein the hyaluronicacid or salt thereof has a molecular weight in the range of between600,000 and 1,800,000.
 7. The hydrogel according to claim 1, wherein thehyaluronic acid or salt thereof has a low average molecular weight inthe range of between 10,000 and 800,000 Da.
 8. The hydrogel according toclaim 1, wherein the hyaluronic acid or salt thereof has a low averagemolecular weight in the range of between 50,000 and 300,000 Da.
 9. Thehydrogel according to claim 1, which comprises an inorganic salt ofhyaluronic acid.
 10. The hydrogel according to claim 9, wherein theinorganic salt of hyaluronic acid is selected from sodium hyaluronate,potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate,magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate.
 11. Acomposition comprising the hydrogel of claim 1 and an active ingredient.12. A composition comprising the hydrogel of claim 1 and apharmacologically active agent.
 13. The composition according to claim12, further comprising a water-soluble excipient.
 14. The compositionaccording to claim 13, wherein the water-soluble excipient is lactose.15. The composition according to claim 12, further comprising apreservative.
 16. A pharmaceutical composition comprising an effectiveamount of the hydrogel of claim 1, together with a pharmaceuticallyacceptable carrier, excipient or diluent.
 17. A pharmaceuticalcomposition comprising an effective amount of the hydrogel of claim 1 asa vehicle, together with a pharmacologically active agent.
 18. Acosmetic article or composition comprising the hydrogel of claim
 1. 19.A sanitary, medical or surgical article comprising the hydrogel ofclaim
 1. 20. A medicament capsule or microcapsule comprising thehydrogel of claim 1.